Heparan sulfate degradation products can associate with oxidized proteins and proteasomes.

• The S-nitrosylated proteoglycan glypican-1 recycles via endosomes where its heparan sulfate chains are degraded into anhydromannose-containing saccharides by NO-catalyzed deaminative cleavage. Because heparan sulfate chains can be associated with intracellular protein aggregates, glypican-1 autoprocessing may be involved in the clearance of misfolded recycling proteins. Here we have arrested and then reactivated NO-catalyzed cleavage in the absence or presence of proteasome inhibitors and analyzed the products present in endosomes or co-precipitating with proteasomes using metabolic radiolabeling and immunomagnet isolation as well as by confocal immunofluorescence microscopy. Upon reactivation of deaminative cleavage in T24 carcinoma cells, [S-35] sulfate-labeled degradation products appeared in Rab7-positive vesicles and co-precipitated with a 20 S proteasome subunit. Simultaneous inhibition of proteasome activity resulted in a sustained accumulation of degradation products. We also demonstrated that the anhydromannose-containing heparan sulfate degradation products are detected by a hydrazide-based method that also identifies oxidized, i.e. carbonylated, proteins that are normally degraded in proteasomes. Upon inhibition of proteasome activity, pronounced colocalization between carbonyl-staining, anhydro-mannosecontaining degradation products, and proteasomes was observed in both T24 carcinoma and N2a neuroblastoma cells. The deaminatively generated products that co-precipitated with the proteasomal subunit contained heparan sulfate but were larger than heparan sulfate oligosaccharides and resistant to both acid and alkali. However, proteolytic degradation released heparan sulfate oligosaccharides. In Niemann-Pick C-1 fibroblasts, where deaminative degradation of heparan sulfate is defective, carbonylated proteins were abundant. Moreover, when glypican-1 expression was silenced in normal fibroblasts, the level of carbonylated proteins increased raising the possibility that deaminative heparan sulfate degradation is involved in the clearance of misfolded proteins.

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MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Läkemedelskemi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Medicinal Chemistry (hsv//eng)

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